title: Synapsin-1 and tau reciprocal O-GlcNAcylation and phosphorylation sites in mouse brain synaptosomes.
abstract: O-linked N-acetylglucosamine (O-GlcNAc) represents a key regulatory post-translational modification (PTM) that is reversible and often reciprocal with phosphorylation of serine and threonine at the same or nearby residues. Although recent technical advances in O-GlcNAc site-mapping methods combined with mass spectrometry (MS) techniques have facilitated study of the fundamental roles of O-GlcNAcylation in cellular processes, an efficient technique for examining the dynamic, reciprocal relationships between O-GlcNAcylation and phosphorylation is needed to provide greater insights into the regulatory functions of O-GlcNAcylation. Here, we describe a strategy for selectively identifying both O-GlcNAc- and phospho-modified sites. This strategy involves metal affinity separation of O-GlcNAcylated and phosphorylated peptides, β-elimination of O-GlcNAcyl or phosphoryl functional groups from the separated peptides followed by dithiothreitol (DTT) conjugation (BEMAD), affinity purification of DTT-conjugated peptides using thiol affinity chromatography, and identification of formerly O-GlcNAcylated or phosphorylated peptides by MS. The combined metal affinity separation and BEMAD approach allows selective enrichment of O-GlcNAcylated peptides over phosphorylated counterparts. Using this approach with mouse brain synaptosomes, we identified the serine residue at 605 of the synapsin-1 peptide, 603QASQAGPGPR612, and the serine residue at 692 of the tau peptide, 688SPVVSGDTSPR698, which were found to be potential reciprocal O-GlcNAcylation and phosphorylation sites. These results demonstrate that our strategy enables mapping of the reciprocal site occupancy of O-GlcNAcylation and phosphorylation of proteins, which permits the assessment of cross-talk between these two PTMs and their regulatory roles.
authors.affiliation: WCU Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology and College of Medicine or College of Pharmacy, Seoul National University, Seoul, Korea.
authors.forename: Min Jueng
authors.initials: MJ
authors.lastname: Kang
authors.forename: Chaeyoung
authors.initials: C
authors.lastname: Kim
authors.forename: Hyobin
authors.initials: H
authors.lastname: Jeong
authors.forename: Byoung-Kyu
authors.initials: BK
authors.lastname: Cho
authors.forename: Ae Lan
authors.initials: AL
authors.lastname: Ryou
authors.forename: Daehee
authors.initials: D
authors.lastname: Hwang
authors.forename: Inhee
authors.initials: I
authors.lastname: Mook-Jung
authors.forename: Eugene C
authors.initials: EC
authors.lastname: Yi
mesh_terms: D000117:Acetylglucosamine / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism*; D002846:Chromatography, Affinity; D006031:Glycosylation; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000302:isolation & purification; D010766:Phosphorylation; D016704:Synapsins / Q000737:chemistry / Q000378:metabolism*; D013574:Synaptosomes / Q000378:metabolism*; D053719:Tandem Mass Spectrometry; D016875:tau Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D010455:Peptides; D016704:Synapsins; D016875:tau Proteins; D000117:Acetylglucosamine